Synthetic oligonucleotides as inducers of erythroid differentiation

ABSTRACT

The invention refers to a synthetic double-stranded oligonucleotide having a length comprised between 10 and 50 bases and a nucleic acid sequence selected from the group consisting of: (a) sequences corresponding to a selected portion of the promoter of human γ-globin gene; and (b) sequences corresponding to a selected portion of the human genomic region comprised between the γ-globin gene and the δβ-cluster, for use as an inducer of erythroid differentiation.

The present invention relates to the use of synthetic oligonucleotideswhich are capable of inducing erythroid differentiation for themanufacture of a medicament for the therapeutic treatment ofβ-thalassemia and neoplastic diseases, and to a pharmaceuticalcomposition including at least one of the said oligonucleotides and apharmaceutically acceptable carrier.

The existence of compounds which are able to induce the synthesis offetal haemoglobin (HbF) in adults is known since long (1–7).

These compounds, herein referred to as “biological response modifiers”,are able to activate the transcription of embryonic and fetal globingenes and to induce erythroid differentiation.

In human adults, the activation of the transcription of γ-globin genesleads to the production of HbF, which mimicks a HPFH (High Persistanceof Fetal Hemoglobin) phenotype; this could reduce the severity ofβ-thalassemia in affected patients (8). Accordingly, recent studies havebeen focused on the search of compounds able to stimulate γ-globin geneexpression at high levels, in an attempt to reduce transfusions inβ-thalassemia patients (12, 13).

In addition, as it has been recently described (9, 10), a combinedtreatment with different biological response modifiers could lead to afurther increase of the expression of γ-globin genes.

It is further known (11) that the treatment of neoplastic cells withcompounds which are able to induce differentiation could be of interestin the therapy of some neoplastic diseases.

The object of the present invention is to find new biological responsemodifiers to be proposed for the treatment of β-thalassemia and/orneoplastic diseases, which exhibit low toxicity in vivo and a high levelof induction of γ-globin gene expression.

Molecules which are able to induce differentiation, exhibiting onlyminor cytotoxic effects in vivo, could in fact reduce side effects whenadministered to patients in clinical trials.

The present inventors have unexpectedly found that double-strandedoligonucleotides having a nucleic acid sequence corresponding toportions of the β-like gene cluster, in particular to some sequences ofthe promoter of the human γ-globin gene and to some sequences comprisedbetween the γ-globin gene and the δβ-gene region, show the saidactivity.

The present invention therefore provides a synthetic double-strandedoligonucleotide having a nucleic acid sequence selected from the groupconsisting of:

-   -   a) sequences corresponding to a selected portion of the promoter        of human γ-globin gene; and    -   b) sequences corresponding to a selected portion of the human        genomic region comprised between the γ-globin gene and the        δβ-cluster,        for use as an inducer of erythroid differentiation.

The nucleic acid sequence of the promoter of human γ-globin is SEQ. IDNO:1 and the nucleic acid sequence of the human genomic region comprisedbetween the γ-globin gene and the δβ-cluster is SEQ. ID NO:2.

Advantageously, the double-stranded oligonucleotide of the invention hasa length comprised between 10 and 50 bases, preferably between 10 and 30bases, and a nucleic acid sequence corresponding to a selected portionof SEQ. ID NO:1 or SEQ. ID NO:2.

Preferably, the double-stranded oligonucleotide of the invention has anucleic acid sequence selected within the portion of SEQ. ID NO:1comprised between positions 220 and 290.

In the present description, the term “oligonucleotide” is meant toinclude also an oligonucleotide wherein the backbone has been modifiedaccording to the approaches commonly used for improvingoligonucleotides' properties such as cellular uptake, target bindingand/or stability. Such modifications include for example themodification of the linkage between the base and/or the sugar moieties.Among the current derivatives and/or techniques available for improvingthe oligonucleotides performances are the conjugation with lipophilicmoieties, chimeric technology, peptide nucleic acids, aptamers.

As it will be further illustrated in the example, it has also been foundthat double-stranded oligonucleotides having a nucleic acid sequence asset forth in SEQ. ID NO:3, SEQ. ID NO:4 (corresponding to selectedregions of the promoter of the human γ-globin gene), SEQ. ID NO:5 andSEQ. ID NO:6 (corresponding to selected regions comprised between thehuman γδβ-globin gene cluster are particularly suitable for inducing ahigh expression of γ-globin genes.

Therefore, the nucleic acid sequence of the double-strandedoligonucleotide of the invention is more preferably selected from thegroup consisting of SEQ. ID No:3, SEQ. ID NO: 4, SEQ. ID No:5 and SEQ.ID NO: 6.

The synthetic oligonucleotides of the invention are able to act bymimicking human γ-globin gene regulatory sequences and by potentiallyinteracting with nuclear proteins, including transcription factors.

The person skilled in the art knows how to obtain a double strandedoligonucleotide; for example it may be obtained by synthesising asingle-stranded oligonucleotide and then specifically annealing thesingle-stranded oligonucleotide with its complementary strand by formingWatson-Crick hydrogen bonds.

The annealing between the two complementary strands may be obtained forexample by incubating the DNA in a solution of 150 mM NaCl at roomtemperature for about two hours. Methods for the synthesis ofsingle-stranded oligonucleotides are found for example in (20).

In one embodiment of the invention, the synthetic double strandedoligonucleotide is used as an inducer of erythroid differentiation incombination with a second biological response modifier, preferablyselected from the group consisting of cytosine arabinoside, retinoicacid, plicamycin, chromomycin, hydroxyurea, guanosine triphosphate(GTP), guanosine diphosphate (GDP), and guanosine monophosphate (GMP).Cytosine arabinoside and retinoic acid are more preferred.

Also within the scope of the invention is the use of a synthetic doublestranded oligonucleotide as previously defined, eventually incombination with a second biological response modifier as previouslydefined, for the manufacture of a medicament for the treatment ofβ-thalassemia and/or neoplastic diseases.

The present invention also provides a pharmaceutical compositioncomprising at least a synthetic double stranded oligonucleotide aspreviously defined, eventually in combination with a second biologicalresponse modifier as previously defined, and a pharmaceuticallyacceptable carrier.

The activity of the double stranded synthetic oligonucleotides of theinvention as inducers of erythroid differentiation has been assessed bydetermining the level of erythroid differentiation induced in a humancultured cell line, as reported in Table 1.

The following example is provided by way of illustration only and is notintended to limit in any way the scope of the invention.

EXAMPLE

The biological activity of the double stranded oligonucleotidesrepresented by SEQ. ID NO: 3, SEQ. ID NO: 4, SEQ. ID NO: 5 and SEQ. IDNO: 6 has been evaluated by determining the ability of theseoligonucleotides to induce erythroid differentiation of the humanerythroleukemia K562 cell line, which is able to undergo erythroiddifferentiation and to increase γ-globin gene expression followingtreatment with a suitable biological response modifier (14–17). Thelevel of erythroid differentiation has been evaluated by thebenzidine-staining (16). The production of haemoglobin has beenevaluated by cellulose acetate gel electrophoresis of cytoplasmicextracts and benzidine staining of the gels (16). The expression ofγ-globin genes has been evaluated by RT-PCR (reverse transcriptase PCR)(18).

These assays have been performed after 6 days of induction with theabove indicated double stranded oligonucleotides.

TABLE 1 Optimal ^(a)Erythroid Oligonucleotide Concentrationdifferentiation (SEQ. ID NOs) (μg/ml) (%) 3 10 55 4 10 40 5 10 57 6 1038 ^(a)Erythroid differentiation = proportion of benzidine-positive K562cells.

In order to analyse hemoglobin production by erythroid induced K562cells, 2 μl of total fresh post-mitochondrial cell lysates wereelectrophoresed on cellulose acetate strips inTris-ethylenediamine-tetraacetic acid (EDTA)-borate buffer. After anelectrophoresis of 30 min at 5 mA, the gels were stained withbenzidine/hydrogen peroxide (1% benzidine in 4.3 M acetic acid, 3% H₂O₂)and photographed. The data obtained show that the Hb produced followingerythroid induction is mainly Hb Portland. Quantitative real-time PCRassay (21) of γ-globin mRNA transcripts was carried out with the use ofgene-specific double fluorescently labeled probes in a 7700 SequenceDetector (PE Biosystems, Warrington Cheshire, UK). The following primerand probe sequences were used for real-time PCR: γ-globin forwardprimer, 5′-TGG CAA GAA GGT GCT GAC TTC-3′ (SEQ. ID NO:7); γ-globinreverse primer, 5′-TCA CTC AGC TGG GCA AAG G-3′ (SEQ. ID. NO:8);γ-globin probe, 5′-FAM-TGG GAG ATG CCA TAA AGC ACC TGG-TAMRA-3′ (SEQ. IDNO:9), where the fluorescent reporter FAM and the quencher TAMRA are6-carboxy fluorescein (FAM) and 6-carboxy-N,N,N′,N′-tetramethylrhodamine(TAMRA) respectively. The results obtained give evidence for an increaseof γ-globin mRNA production. For instance, the oligonucleotidecorresponding to SEQ. ID NO:3 induced a nine-fold increase of γ-globinmRNA production with respect to uninduced K562 cells.

The high level of biological activity and the expected low level of invivo cytotoxicity of the oligonucleotides of the invention allow topropose these molecules as promising candidates for use in thedevelopment of pharmacological approaches for the treatment ofβ-thalassemia and/or neoplastic diseases.

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1. A synthetic double-stranded oligonucleotide of 15–50 bases whereinthe synthetic double-stranded oligonucleotide comprises the sequence ofSEQ. ID NO.
 3. 2. A pharmaceutical composition comprising at least thesynthetic double-stranded oligonucleotide according to claim 1 and apharmaceutically acceptable carrier.
 3. A pharmaceutical compositioncomprising at least the synthetic double-stranded oligonucleotideaccording to claim 1, a further modifier of a biological response and apharmaceutically acceptable carrier.
 4. The pharmaceutical compositionaccording to claim 3, wherein said further modifier of the biologicalresponse is plicamycin.
 5. The synthetic double-stranded oligonucleotideaccording to claim 1, in combination with a further biological responsemodifier selected from the group consisting of cytosine arabinoside,retinoic acid, plicamycin, chromomycin, hydroxyurea, guanosinetriphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate(GMP).
 6. A method of therapeutic treatment, comprising: administering asynthetic double-stranded oligonucleotide of 15-50 bases to a patient,wherein said synthetic double-stranded oligonucleotide comprises thesequence of SEQ. ID NO.
 3. 7. The method of therapeutic treatment ofclaim 6, further comprising: administering a pharmaceutically acceptableto carrier to the patient.
 8. The method of therapeutic treatment ofclaim 7, further comprising: administering a further modifier ofbiological response to the patient.
 9. The method of therapeutictreatment of claim 8, wherein said further modifier of biologicalresponse is plicamycin.